1. Field of the Invention
The present invention relates to a method and a device for measuring distributions and changes of concentrations of intracellular ions which contribute to controls, etc. of cell functions.
2. Related Background Art
Fluorescent probe dyes, such as Fura-2, Indo-1, etc., are used to determine concentrations of ions, such as calcium ions, etc. present in live cells. Methods for synthesizing these dyes, and characteristics of these dyes are described in the following references, "The Journal of Biological Chemistry (1985), Vol. 260, p. 3340-3450" and "Biophysical Journal, Vol. 54, (1988), p. 1089-1104". These probe dyes have the property that their fluorescence characteristics change depending on bonds and dissociations of specific ions. To measure intracellular ion concentrations by using such property, the probe dyes are introduced into cells, and intensities of fluorescence generated by an excitation beam of certain wavelength are measured.
But the measurement based on an excitation beam at only one wavelength and fluorescence of only one wavelength cannot give accurate measured values when an concentration of probe dyes in the cell are unknown or a distribution of probe dyes in the cell is disuniform. There is a risk that changes of fluorescence intensities which do not depend on ion concentrations but caused by decreases of the fluorescence may be also measured.
As a method for removing this risk, fluorescence intensities for excitation beams of two different wavelengths, and intensities of fluorescence of two different wavelengths for one kind of an excitation beam are respectively measured, and their respective ratios are measured.
This method is described in good detail in Atsuo Miyakawa et al., "Bunseki Kagaku, Vol. 38, No. 11, p.642-p.649 (1989)", and "Photomedicine and Photobiology, Vol. 13, p.15-p.19 (1991)".
The above-described dual wavelength fluorescence measuring method is usable for systems to only a certain ion of which the probe dyes is reactive, but it is considered that the probe dyes bond with proteins and components other than ions, which are present in high concentrations in cells. Accordingly fluorescence emission spectra and chelate constants are adversely changed. This dual wavelength fluorescence measuring method cannot correct errors due to interactions between the dyes and components other than ions. Accurate ion concentrations cannot be determined.